Microarray technology is readily used in biological research as it provides unprecedented information on nucleic acids in a wide range of applications such as gene expression and genotyping (Ramsay, 1998, Nature Biotechnology 16: 40-44; U.S. Pat. No. 5,837,832). Like DNA, RNA microarrays have also emerged as combinatorial tools as a result of the increasing interest in the use of RNAi and RNA aptamers. The fabrication of such microarrays has evolved from spotting cDNA onto filter paper to more advanced methods such as photolithography with masks or micromirror arrays. While microarrays may be fabricated through immobilization or spotting of pre-synthesized oligonucleotides, the in situ synthesis of DNA microarrays has become the preferred technique as it provides unparalleled chip complexity in an efficient and cost effective manner. However, unlike DNA, the construction of RNA microarrays is limited to spotting as they are far more challenging to synthesize in situ. In addition RNA is more susceptible to enzymatic and chemical hydrolysis.
In general, unlike DNA, there are challenges associated with synthesizing RNA oligonucleotides as a result of the distinct 2′-hydroxyl group present in RNA. The 2′-hydroxyl must be appropriately protected in order to prevent phosphodiester bond isomerization or degradation and to allow for efficient monomer coupling during oligonucleotide synthesis (Reese, 2005, Org. Biomol. Chem. 3: 3851-3868). To date, there have been many attempts to design protecting groups that embody the conditions required for the construction of high quality oligoribonucleotides. The most widely used 2′-protecting group is the 2′-O-t-butyldimethylsilyl (TBDMS) group, introduced in the oligonucleotide area by Ogilvie et at., 1974, Tetrahedron Lett. 15: 2861-2867. This protecting group is removed at the end of RNA chain assembly by fluoride ions. Other silyl protecting groups such as 2′-O-TOM (2′-O-triisopropylsilyloxymethyl) have been used in the synthesis of RNA (Pitsch et al., 1999, Helv. Chim. Acta 82: 1753-1761). Alternate protecting groups are the photolabile group 2′-(2-nitrophenyl)ethoxycarbonyl, 2′-(2-nitrophenyl)ethylsulfonyl and 2′O-(O-nitrobenzyl) substituents and the acid labile acetals such as the 2′-tetrahydropyranyl, 2′-O-Fpmp (1-(2-fluorophenyl)-4-methoxypiperidin-4-yl), 2′-O-Cpep (1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl), 2′-O-4-MABOM (2′-O-[4-(N-methylamino)benzyloxy]methyl, and 2′-ACE (2′-O-bis(2-acetoxyethoxy)methyl). Synthesis of 3′- and 5′-O-levulinyl-2′-deoxy- and 2′-O-alkylribonucleosides has been described by Javier et al., 2003, Tetrahedron 14: 3533-3540. The levulinyl group has been employed for protection of the 5′-hydroxyl group in the synthesis of oligoribonucleotides by the phosphoramidite approach (Iwai and Ohtsuka, 1988, Nucleic Acids Res. 16: 9443-9456). In all cases the synthesis of oligoribonucleotides is an elaborate multistep process, which entails assembly of the oligonucleotide chain, deprotection of the base labile nucleobase protecting groups, cleavage from the support, followed by removal of the 2′-hydroxyl protecting group.
At present there is a dearth of reports on the fabrication of RNA microarrays in the literature. Generally RNA microarrays are synthesized through immobilization of a pre-synthesized RNA strand in its native form, which requires expensive synthesis and purification of modified RNA (i.e., thiol, biotin or amino terminated end), which subsequently limit chip complexity. In addition, such methods leave RNA oligonucleotides vulnerable to RNA degradation as they are in the deprotected form. An alternative strategy uses surface RNA-DNA ligation chemistry to create RNA microarrays from 5′-phosphate modified DNA microarrays. This strategy involves expensive and elaborate procedures that are limited by reliability and complexity. There are no examples in the literature of an in situ synthesis of RNA microarrays.
RNA interference (RNAi) therapeutics represents a fundamentally new way to treat human diseases (Manoharan, 2004, Curr. Opin. Chem. Biol. 8: 570-579). However, achieving targeted tissue and cellular delivery, stabilization in vivo, and cost effective large scale synthesis of RNA are significant bottlenecks in the development of RNAi technology.
There is a need to develop synthetic strategies that permits both the growth and deprotection of RNA chains that remain attached to a solid polymer support or to a glass or chip surface. The present invention addresses these and related needs.